Surface Of Chronic Lymphocytic Leukemia Cells Research Articles

Definition and Characterization of a Peptide Warehouse for the Patient-Individualized Peptide Vaccination Study (iVAC-L-CLL01) after First Line Therapy of CLL

The treatment of chronic lymphocytic leukemia (CLL) has substantially advanced over the last decade with chemo/immunotherapy as standard of care achieving overall response rates up to 90%. However, besides allogeneic stem cell transplantation, so far no curative treatment for CLL is available. This is mostly due to the persistence of residual CLL cells after therapy (minimal residual disease, MRD), which cause disease relapse. Thus, novel strategies such as peptide-based immunotherapy aiming to eliminate MRD are needed to achieve lasting disease remission or even cure. With this aim, we implemented a first-in-man multi-centric clinical phase II study (iVAC-L-CLL01, NCT02802943), in which patient-individualized peptide vaccination with or without lenalidomide as systemic immune modulator is applied after first-line therapy of CLL. The peptide vaccine cocktail consists of 10 tumor-associated peptides selected according to the HLA ligandome of the individual patients from a pre-manufactured “peptide warehouse”. This peptide warehouse contains CLL-associated antigens, which are frequently and exclusively presented by HLA molecules on the surface of CLL cells. The peptides have been selected using a direct mass spectrometry based comparative analysis of the HLA ligandome of 30 CLL patients and a normal tissue ligandome database (Kowalewski et al. PNAS, 2015). The warehouse includes 35 HLA class I and 5 HLA class II peptides. The HLA class I peptides are restricted by the 7 most common HLA class I allotypes (A*01, A*02, A*03, A*24, B*07, B*08 and B*44). Prior to clinical application, the immunogenicity of the warehouse peptides was validated using different T-cell assays. We performed IFNg ELISpot assays using PBMC obtained from CLL patients and healthy donors. Peptide-specific memory T-cell responses were detected for 20/35 (57%) HLA class I peptides (A*01, A*02, A*03 and B*08) and 5/5 (100%) HLA class II peptides in samples obtained from CLL patients. Immune responses in healthy donors could not be detected for any of the 40 warehouse peptides, indicating a strictly CLL directed T-cell response. The immunogenicity of the remaining peptides could not be approved by detection of spontaneous memory T-cell responses in CLL patients due to lack of HLA-matched CLL-patient samples. Hence, we implemented artificial APC-based in vitro priming assays using CD8+ T cells of healthy donors proofing the ability of the peptides to prime naïve CD8+ T cells. For all 15 analyzed peptides (HLA A*24, B*07 and B*44), an induction of tetramer positive CD8+ T cell populations with frequencies of 0.1-35.1% could be detected. Therefore, all 40 warehouse peptides could be confirmed as immunogenic T-cell epitopesThe iVAC-L-CLL01 study is recruiting since October 2016. Vaccination of the first included study patient was performed in July 2017. DisclosuresNo relevant conflicts of interest to declare.

Obinutuzumab Activates CD64 More Potently Than Other Anti-CD20 Antibodies in Chronic Lymphocytic Leukemia (CLL)

IntroductionTreatment with monoclonal antibodies has revolutionized clinical medicine, especially in the fields of cancer and immunology. One of the oldest antibodies, which is widely used for the treatment of lymphomas and autoimmune diseases, is the anti-CD20 antibody rituximab. In recent years, new antibodies against CD20 have been developed, notably ofatumumab and obinutuzumab. Both antibodies are currently used for the treatment of chronic lymphocytic leukemia (CLL), with a possible clinical advantage of obinutuzumab over rituximab. In addition to CLL, obinutuzumab was recently approved for the treatment of relapsed/refractory follicular lymphoma in patients who had previously received a rituximab-containing regimen.An important mechanism of action of therapeutic monoclonal antibodies is activation of immune cells via Fc receptors (FcγRs). Surprisingly, however, the interactions of various therapeutic antibodies with various Fc receptors have not been fully elucidated. Moreover, the available data on the interaction of different anti-CD20 antibodies and human Fc receptors is based on affinity measurements, mainly surface plasmon resonance (SPR), and not on functional assays. Although primary immune cells can help us gain information regarding the activation of Fc receptors, these cells are difficult to handle and usually express several Fc receptors.MethodsHere we established a functional assay to study how different anti-CD20 antibodies activate different human Fc receptors. To this aim, we stably transfected mouse BW5147 cells (BW cells) with chimeric FcγRs which included the extracellular part of a given FcγR fused to the transmembrane and cytoplasmic segments of the mouse CD3ζ chain. Activation of a specific FcγR results in secretion of mouse interleukin-2 (mIL-2) which can be detected by enzyme-linked immunosorbent assay (ELISA). We prepared BW cells which express the following human FcγR-CD3ζ chimeras: CD16A (FcγRIIIA, low and high affinity variants 158F and 158V respectively), CD32A (FcγRIIA, low and high affinity variants, 131R and 131H respectively), and CD64 (FcγRI). The advantages of this system are that it is a functional assay, easy to use, sensitive (was used to detect anti-viral antibodies in patients) and enables to study the activation of a single Fc receptor. Additionally, the activation of a specific Fc receptor by this reporter system is studied in the context of the natural ligand of the antibody, which better reflects the activity of the antibody in vivo .We assessed the activation of Fc receptors by different anti-CD20 antibodies which were incubated with chronic lymphocytic leukemia (CLL) cells obtained from patients.ResultsUsing this functional assay we corroborated previous reports regarding the higher affinity of obinutuzumab to CD16A. Importantly, we discovered that obinutuzumab activates CD64 significantly more efficiently than rituximab or ofatumumab. We show that the advantage of obinutuzumab in activating CD64 translates into better elimination of CLL cells by primary macrophages. Mechanistically, we found that CD64 is activated more potently by obinutuzumab since obinutuzumab does not internalize when bound to its target protein CD20, in contrast to rituximab and ofatumumab. We show that the internalization of rituximab and ofatumumab is mediated by binding to the FcγR CD32B, which is expressed on the surface of CLL cells. Indeed, blocking of CD32B led to increased activation of CD64 by rituximab and ofatumumab.DiscussionUsing a functional assay we found that obinutuzumab activates CD64 more potently than other anti-CD20 antibodies. Is it possible that this advantage is relevant only to tumor cells with high expression of CD32B. This could be applied clinically by incorporating obinutuzumab instead of rituximab in certain lymphoproliferative diseases (e.g., CLL) or specifically in patients with high expression of CD32B. Furthermore, the BW reporter assay can be applied to other therapeutic antibodies and may assist in developing antibodies with improved immunological properties. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Research progress of chimeric antigen receptor T cell immunotherapy for chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a chronic B lymphocyte clonal proliferative disease with a median age at onset of more than 70 years. The clinical manifestations are CD5 and CD19 double positive mature small lymphocytes abnormal aggregation in peripheral blood, bone marrow, spleen and lymph nodes, which cause corresponding clinical symptoms. In recent years, targeted drugs such as CD20 monoclonal antibodies, B cell lymphoma (BCL)-2 inhibitors, Bruton tyrosine kinase inhibitor (BTKi), and allogeneic hematopoietic stem cell transplantation (allo-HSCT) continues to appear, the efficacy of CLL continues to improve. But the case fatality rate of patients with relapsed/refractory CLL remains high. In the era of immune cell therapy, chimeric antigen receptor T cell (CAR-T) immunotherapy achieves specific recognition of CLL cell surface antigen and precise target killing of tumor cells. This article reviews the research of CAR-T immunotherapy in CLL. Key words: Leukemia, lymphocytic, chronic, B-cell; Immunotherapy; Recurrence; Antigens, CD19; Chimeric antigen receptor T-cell

Siglec-6 on Chronic Lymphocytic Leukemia Cells Is a Target for Post-Allogeneic Hematopoietic Stem Cell Transplantation Antibodies.

Although the 5-year survival rate of chronic lymphocytic leukemia (CLL) patients has risen to >80%, the only potentially curative treatment is allogeneic hematopoietic stem cell transplantation (alloHSCT). To identify possible new monoclonal antibody (mAb) drugs and targets for CLL, we previously developed a phage display-based human mAb platform to mine the antibody repertoire of patients who responded to alloHSCT. We had selected a group of highly homologous post-alloHSCT mAbs that bound to an unknown CLL cell surface antigen. Here, we show through next-generation sequencing of cDNAs encoding variable heavy-chain domains that these mAbs had a relative abundance of ∼0.1% in the post-alloHSCT antibody repertoire and were enriched ∼1,000-fold after three rounds of selection on primary CLL cells. Based on differential RNA-seq and a cell microarray screening technology for discovering human cell surface antigens, we now identify their antigen as Siglec-6. We verified this finding by flow cytometry, ELISA, siRNA knockdown, and surface plasmon resonance. Siglec-6 was broadly expressed in CLL and could be a potential target for antibody-based therapeutic interventions. Our study reaffirms the utility of post-alloHSCT antibody drug and target discovery. Cancer Immunol Res; 6(9); 1008-13. ©2018 AACR.

The Oxazolidinone Derivative Locostatin Induces Apoptosis in CLL Cells through Inhibition of AKT and MAPK-ERK1/2 Signaling Under Conditions That Mimic the Tumor Microenvironment

BACKGROUND The Raf-1/MEK/MAPK-ERK1/2 pathway plays an important role in the pathogenesis and progression of multiple forms of cancer, including Chronic Lymphocytic Leukemia (CLL). Recent pre-clinical trials of the Raf-1 inhibitor Sorafenib demonstrate that targeting this pathway may represent a therapeutic option for CLL.Raf-1 activity is negatively regulated by the Raf-1 kinase inhibitory protein (RKIP). However, in CLL cells evidence suggests that phosphorylation of RKIP by protein kinase C (PKC) may restrict RKIP binding to Raf-1 and promote its binding and inhibition of G-coupled protein receptor 2 (GRK2). Consequently, GRK2, which would normally bind AKT and MEK1/2, would no longer suppress activity of these proteins. It is conceivable, therefore, that deregulation of RKIP may contribute to the over activity of the Raf-1 and AKT-mediated pathways that has been described in freshly isolated CLL cells.METHODS We sought to determine the effects of inhibiting RKIP on the survival and migratory capacity of CLL cells in vitro by using Locostatin, a specific inhibitor of RKIP binding to Raf-1 and GRK2. The proportion of apoptotic primary CLL cells following Locostatin treatment in either media alone or in the presence of a supportive stroma was assessed by flow cytometry. The mechanisms of action of Locostatin were investigated by western blotting for the phosphorylated forms of AKT and ERK1/2.RESULTS Locostatin induced a dose dependent decrease in the proportion of viable cells from CLL patients (n = 6) and a panel of haematological lines (n = 8). The IC50 for Locostatin was significantly (P < 0.001) lower against primary CLL samples analysed (11.14 +/- 4.86 µM) than in peripheral blood mononuclear cells from healthy volunteers (72.71 +/- 9.85 µM). Locostatin was also effective against CLL cells cultured in the presence of a CD40L-expressing fibroblast layer.Locostatin treatment reduced the expression of the chemokine receptor CXCR4 on the CLL cell surface with a concomitant decrease in their migration along a stroma-derived factor 1α (SDF-1α) gradient and significantly reduced the proportion of cycling CLL cells following induction by CpG oligonucleotide/IL2 (Dsp30/IL-2).We demonstrate by western blotting that RKIP is phosphorylated on S153 in CLL patient samples (Figure 1), supporting our hypothesis that RKIP may not be able to bind Raf-1 in these cells. Furthermore, we show that Locostatin treatment inhibits phosphorylation of AKT (S473) and ERK1/2 in CLL cells cultured in media alone or on CD40L fibroblasts (Figure 2).CONCLUSIONS These data suggest that deregulation of RKIP in CLL cells may contribute to their survival and proliferation by increasing the activities of AKT and ERK1/2. We show that Locostatin, a specific RKIP inhibitor, down-regulates the activity of both AKT and ERK1/2 in CLL cells cultured in media or a model of the tumor microenvironment, decreases cell viability and reduces the migratory and proliferative capacities of the leukemic cells. Our data suggest that RKIP may represent a novel target for therapy in CLL, which can be effectively inhibited by Locostatin. [Display omitted] [Display omitted] DisclosuresMulligan:Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi Aventis: Research Funding; Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria.

Nurse-like Cells Engage Sigm and Sigd on Chronic Lymphocytic Leukemia (CLL) Cells: Implications for BCR Signaling Activation and Functional Outcome

Introduction: Nurse-like cells (NLCs) are found in secondary lymphoid organs of Chronic Lymphocytic Leukemia (CLL) patients and represent an important component of the CLL microenvironment. NLCs can differentiate in vitro from monocytes after coculture of CLL cells with peripheral blood mononuclear cells and support the survival and proliferation of CLL cells through several mechanisms, including BCR signaling activation and CCL3/4 chemokine production.Aim: The aim of the present study is to gain insight on the mechanism of BCR signaling activation in CLL-NLC coculture with a focus on the functional differences between IgM and IgD signaling.Methods: BCR surface expression was analyzed by flow cytometry; CLL cells were stimulated with soluble anti-IgM or anti-IgD and 48-hour cell viability was evaluated by flow cytometry; CCL3/4 chemokine secretion was analyzed after 24-hour anti-IgM or anti-IgD stimulation by ELISA.Results: We analyzed the levels of surface IgM (sIgM) and IgD (sIgD) expression on CLL cells before and after coculture with NLCs. Rapid downmodulation of both sIgM and sIgD was observed in CLL-NLC cocultures (Figure 1). CLL cells carrying unmutated IGHV genes (U-CLL) expressed significantly higher sIgM and sIgD than mutated CLL (M-CLL) (p(sIgM)<0.0001; p(sIgD)=0.0008; 35 samples/group) and downmodulated both receptors to a greater extent than M-CLL in the presence of NLCs (p(sIgM)=0.04; p(sIgD)=0.0047; 7 samples/group). Downmodulation of both sIgM and sIgD was not observed on CLL cells (6 patients) after coculture with the human mesenchymal stromal cell line hTERT, suggesting that receptor downregulation is a direct effect of the CLL-NLC interaction. Antigen stimulation is required for receptor internalization, as rapid downmodulation of both sIgM and sIgD was observed following BCR stimulation with soluble anti-IgM and anti-IgD in 14 samples.Two outcomes of the CLL-NLC interaction are protection of CLL cells from apoptosis and CCL3/4 chemokine production. We replaced NLCs with anti-IgM and anti-IgD and examined the contribution of IgM and IgD signaling in mediating CLL cell survival and CCL3/4 secretion. IgM stimulation induced significantly higher protection from in vitro apoptosis as compared to IgD (p=0.03), as analyzed on 18 samples. In addition, U-CLL cells were more responsive to IgM-induced protection from apoptosis as compared to M-CLL (p=0.04, 9 samples/group). Both CCL3/4 were produced exclusively after anti-IgM stimulation, in particular of U-CLL; surprisingly, no CCL3/4 chemokine production was detected after anti-IgD stimulation, as analyzed on 12 samples. Simultaneous ligation of both IgM and IgD receptors did not affect viability and chemokine production, as compared to IgM stimulation alone.Conclusion: Similar to what is observed when CLL cells are stimulated with soluble anti-IgM and anti-IgD, NLCs induce sIgM and sIgD downregulation on the surface of CLL cells, which is indicative of antigen engagement by the BCR. This effect appears to be NLC-specific, as it is not observed when CLL cells are co-cultured with the human mesenchymal stromal cell line hTERT. In vitro stimulation of CLL cells with anti-IgM induces greater protection from apoptosis and CCL3/4 chemokine production than anti-IgD stimulation, in particular in the U-CLL subset. Taken together, these results suggest that NLCs may present antigens to CLL cells, leading to IgM and IgD receptor engagement and downregulation. In this setting, IgM stimulation may be more relevant than IgD in the induction of NLC-specific protective effects, including CLL-cell survival and CCL3/4 production. Deeper insight into the nature of the CLL-NLC interaction and of the antigen(s) involved will provide a better understanding of NLC-mediated BCR triggering in CLL subsets, with therapeutic implications in the context of microenvironment or BCR signaling interference. [Display omitted] DisclosuresO’Brien:Amgen, Celgene, GSK: Consultancy; CLL Global Research Foundation: Membership on an entity’s Board of Directors or advisory committees; Emergent, Genentech, Gilead, Infinity, Pharmacyclics, Spectrum: Consultancy, Research Funding; MorphoSys, Acerta, TG Therapeutics: Research Funding.

Obinutuzumab may chart the way to improved QOL for CLL patients.

Obinutuzumab recently received accelerated approval from the US Food and Drug Administration with breakthrough therapy designation for use in combination with chlorambucil in patients with untreated chronic lymphocytic leukemia (CLL). Obinutuzumab is a CD20 targeting fully humanized, type II, IgG1 antibody. CD20 is weakly expressed on the surface of CLL cells but has been demonstrated to be an effective in vivo target as shown by the activity observed with the use of rituximab and ofatumomab.2-4 Obinutuzumab binds selectively to the extracellular domain of CD20 with reduced internalization and its structural modifications explain its enhanced effectiveness. The antibody is modified in the hinge region which allows for more potent direct cytotoxicity. More importantly, afucosylation in the Fc region allows for enhanced antibody dependent cellular cytotoxicity (ADCC) through robust engagement of Fc-gamma receptor type III on effector cells. Together, these modifications translate into a higher efficacy compared with rituximab both in preclinical and clinical studies.

Matriptase Is Highly up-Regulated in Chronic Lymphocytic Leukemia and Promotes Cancer Cell Invasion

AbstractAbstract 4612Proteases are known to play a critical role in cancer development and progression. Matriptase is an epithelial transmembrane serine protease essential for skin development and barrier function. Matriptase overexpression has been reported in many human solid tumors including prostate, breast, ovarian, kidney and lung cancers, indicating that matriptase may play an important role in cancer. In mice, dysregulated matriptase activity promotes malignant transformation and carcinogenesis.In human blood, low levels of matriptase mRNA expression were detected in monocytes and B-lymphocytes but the biological significance is unclear. In this study, we examined matriptase mRNA and protein expression in 15 hematological cancer cell lines and 223 bone marrow samples from patients with leukemia by reverse transcription PCR (RT-PCR), quantitative real-time PCR (qRT-PCR) and Western blotting. Flow cytometry and immunostaining were used to determine the cell membrane expression of matriptase in B cell-derived Namalwa cells and bone marrow cells from leukemia patients. Small hairpin RNA (shRNA) interference and a recombinant matriptase inhibitor, hepatocyte growth factor activator inhibitor (HAI)-1, were used to inhibit matriptase expression and activity in Namalwa, Raji and chronic lymphocytic leukemia (CLL) cells to test the effect of matriptase inhibition on cancer cell invasion in matrigel-based assays.Our results showed that matriptase mRNA was highly expressed in Namalwa and Raji cells and in bone marrow cells from patients with CLL. Such an expression profile was not found in bone marrow cells from patients with acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). By Western blotting, matriptase protein expression was confirmed in bone marrow samples from the majority (79/81, 97.5%) of CLL patients. By immunostaining and flow cytometry, matriptase protein was localized on the surface of Namalwa and CLL cells. In these cells, the matriptase inhibitors, HAI-1 and HAI-2, were not detected, indicating that the upregulated matriptase activity may not be inhibited in CLL cells. In functional studies, silencing matriptase gene expression by specific shRNAs markedly reduced Namalwa and Raji cells and bone marrow cells from CLL patients in invading matrigels. Similarly, a recombinant HAI-1 dose-dependently inhibited matrigel invasion of these cancer cells.In conclusion, our results showed that matriptase was highly expressed on the surface of CLL cells. Inhibition of matriptase by gene silencing or specific inhibitor prevented CLL cells in invading matrigels, indicating that matriptase contributes to the invasiveness of the cancer cells. Our results suggest that matriptase may represent a new cell surface marker for CLL and that blocking matriptase expression or activity may be used as a therapeutic strategy to inhibit CLL progression. Disclosures:No relevant conflicts of interest to declare.

Generation of a Platform for Identification of CLL Specific Cell Surface Proteins Targeted by Anti-Tumor Antibodies in Patient Sera After Allogeneic Hematopoietic Cell Transplantion

AbstractAbstract 1349 Background:Clinical and translational studies suggest that allogeneic hematopoietic cell transplantation (alloHCT) may induce production of anti-tumor antibodies in the recipient after transplantation. We hypothesized that this phenomenon can serve as a platform for the generation of novel therapeutic monoclonal antibodies (mAbs). An important step to this goal is the identification of targeted antigens with defined ability to support antibody induced cell death. To address this challenge, we developed a chronic lymphocytic leukemia (CLL) membrane protein display system capable of discovering cell surface proteins targeted by serum antibodies and applied this tool to post-alloHCT patient samples. Methods:Total and mRNA was purified from peripheral blood mononuclear cells from six untreated CLL patients and used to generate cDNA. The patient cDNA was pooled, cloned into the retroviral vector pBMN, and expressed in the murine T-cell line Bw 5147 (Bw-CLL-Lib). Enrichment of Bw-CLL-Lib cells displaying membrane proteins of interest was performed via fluorescence activated cell sorting. The unselected Bw-CLL-Lib pool was blocked with recombinant human Fc followed by staining with 1:200 diluted post-alloHCT patient sera and by secondary staining with pooled Alexa Fluor 647 labeled goat-anti-human-lambda and -anti-kappa light chain specific antibodies. Bw-CLL-Lib cells positively binding to serum antibodies were collected and re-grown in culture to 1×106 cells. A second round of enrichment was performed, after which the Bw-CLL-Lib cells were placed in limiting dilution culture. Individual clones were screened for serum reactivity and the retroviral inserts in reactive Bw-CLL-Lib clones were rescued via PCR and sequenced. Proteins of interest were re-expressed in Bw 5147 cells and used to confirm reactivity of the patient serum with specific cell surface proteins. Results:The Bw-CLL-Lib cell pool was screened separately with serum from ten patients with CLL post unrelated donor alloHCT. Serum from three patients enriched positively binding Bw-CLL-Lib clones. Thus far, we have successfully identified serum antibodies to a membrane proximal epitope on a therapeutically relevant CLL cell surface protein. Conclusions:Here we demonstrate a methodology for identifying targets of anti-tumor antibodies in serum from patients after alloHCT. This technique yields a high degree of successful identification of antibody reactivity with cell surface proteins in post alloHCT serum samples. When combined with post-alloHCT antibody Fab phage display (Baskar et al., Blood 114, 4494–4502, 2009) this methodology forms a complete drug and target discovery platform for the generation of tumor specific mAbs derived from alloHCT patients. Disclosures:No relevant conflicts of interest to declare.

Identification of Novel Tumor-Associated Antigens for Chronic Lymphocytic Leukemia (CLL) Based On HLA Ligandome Analysis – New Targets for Peptide Based Immunotherapy

AbstractAbstract 4119Recurrent disease, selection for chemo resistant clones and inhibition of immune effector functions are limitations of chemotherapeutic treatment of cancer including CLL. This is even more since graft-versus-leukemia effects and remissions after donor lymphocyte infusions have been correlated to long-term CLL free survival. Clonotype analysis of such cases suggested clonally expanded CD8+ T cells that recognize tumor associated antigens (TAAs) presented on HLA (human leukocyte antigen) as mediators of the observed effects, thus making CLL an attractive target for peptide vaccine based immunotherapy. For this goal we established the approach of direct isolation and identification of naturally processed and presented HLA ligands from tissues of interest by affinity chromatography and mass spectrometry. Comparative, semi-quantitative analysis of the HLA ligandomes of malignant and benign samples provided the rationale for selection of potential targets. 42 CLL patients (ages 48–85; median 70 years) of different HLA types and disease stages (Binet A, 24 patients; Binet B, 11 patients; Binet C, 7 patients) were enrolled in this study. Furthermore we collected blood samples from 50 healthy volunteers. HLA class I ligands were isolated from CLL cells as well as benign B cells from healthy donors and unsorted healthy PBMC using a standard affinity chromatography protocol implementing the pan-HLA class I specific antibody W6/32. Liquid chromatography coupled mass spectrometry (LC-MS/MS) based peptide analysis was performed on an LTQ Orbitrap hybrid mass spectrometer followed by annotation of fragment spectra using the MASCOT search algorithm against the human proteome comprised in the SwissProt database. Spectral counting based analysis provided semi-quantitative information regarding the abundance of HLA ligands and their source proteins in the respective ligandomes. In addition, HLA quantification experiments on the cell surface of CLL cells and autologous healthy B cells were performed using a flow cytometric indirect immunofluorescence assay. For this interim analysis we completely analyzed the HLA ligandomes of 7 CLL patients and 10 healthy controls. In total, we identified more than 15.000 different HLA ligands representing more than 7300 different proteins. This comprised more than 6,500 different ligands from CLL cells representing a total of 4,149 source proteins. Comparative analysis of representation in the HLA ligandomes of CLL cells, healthy B cells and healthy PBMC identified 1741 different source proteins as being exclusively expressed in CLL. Semi-quantitative evaluation revealed 138 of these proteins as being highly expressed on CLL (e.g. SET proto-oncogene, Pim-1 Oncogene, Mucin 1). Flow cytomerty based quantification of surface HLA expression revealed similar amounts of HLA class I (p=0.23, unpaired t-test) and II (p=0.33, unpaired t-test) molecules on CLL cells and autologous benign B lymphocytes, with a trend to higher HLA expression on CLL cells.Taken together, the presented strategy enabled the identification of a vast array of both known and novel TAAs and their corresponding naturally processed and presented HLA ligands in CLL. It pinpointed highly overrepresented TAAs for further analysis and immunogenicity testing. Expansion of the dataset after analysis of all enrolled patients will provide an unprecedented in-depth characterization of the HLA ligandome of CLL for future immunotherapeutic approaches. Disclosures:No relevant conflicts of interest to declare.

Programmed Death-1 and Its Ligand Are Novel Immunotolerant Molecules Expressed on Leukemic B Cells in Chronic Lymphocytic Leukemia

Programmed death-1 (PD-1) is an immunoreceptor predominantly expressed on exhausted T cells, which through an interaction with its ligand (PD-L1), controls peripheral tolerance by limiting effector functions of T lymphocytes. qRT-PCR for PD-1, PD-L1 and their splicing forms as well as flow cytometric assessment of surface expression was performed in a cohort of 58 chronic lymphocytic leukemia (CLL) patients. In functional studies, we assessed the influence of the proliferative response of leukemic B-cells induced by IL-4 and CD40L on PD-1 transcripts and expression on the protein level. The median level of PD-1, but not PD-L1, transcripts in CLL patients was higher in comparison to healthy volunteers (HVs, n = 43, p = 0.0057). We confirmed the presence of PD-1 and PD-L1 on the CLL cell surface, and found the expression of PD-1, but not PD-L1, to be higher among CLL patients in comparison to HVs (47.2% vs. 14.8%, p<0.0001). The Kaplan-Meier curves for the time to progression and overall survival in groups with high and low surface expression of PD-1 and PD-L1 revealed no prognostic value in CLL patients. After stimulation with IL-4 and CD40L, protein expression of PD-1 was significantly increased in samples that responded and up-regulated CD38. PD-1, which is aberrantly expressed both at mRNA and cell surface levels in CLL cells might represent a novel immunotolerant molecule involved in the pathomechanism of the disease, and could provide a novel target for future therapies.

Fludarabine, Cyclophosphamide, and Alemtuzumab (FCC) In Relapsed/Refractory Patients with B-Cell Chronic Lymphocytic Leukemia (CLL): Final Report of the Italian Study

AbstractAbstract 1384 Introduction:Purine Analogues in combination with cyclophosphamide (C) have a relevant advantage over monotherapy approach in patients(pts) with CLL. However, pts in complete remission (CR) eventually relapse and require treatment, demonstrating the need for improved treatments able to induce “better quality” and thus more durable responses. Monoclonal antibody alemtuzumab (CAM), directed towards CD52 antigen expressed on the surface of CLL cells has been proven to be the most effective antibody in CLL treatment. As previously reported by Elter et al. (JCO 23:7024-7031.2005) CAM appears to have synergistic activity with fludarabine (F) in vivo in relapsed and refractory CLL. Therefore, we designed a phase II study to determine the efficacy and safety of a 4-weekly combination regimen consisting of F, C, and CAM in CLL with relapsed or refractory disease after at least 1 line of treatment. Methods:The FCC regimen consisted of oral F 40 mg/m2/day and oral C 250 mg/m2/day immediately followed by sc CAM 10 mg/day (Days 1–3). A-2 day escalation of CAM was administered prior to the first cycle. This combination was repeated on Day 29 for up to 6 cycles. According to the safety profile of the schedule described above the dose of CAM was increased after the first cohort of 10 treated patients from 10 mg to 20 mg. Minimal residual disease (MRD) was measured by 4-color flow cytometry.Anti-infective prophylaxis included acyclovir 400 mg twice daily and trimethoprim sulfamethoxazole (TMP + SMX) 1000 mg given every other day from treatment initiation until 6 months after treatment end. CMV antigen in blood was tested in 7–days interval. Results:Recruitment was stopped when the planned number of 43 patients have been enrolled in the trial. The median age of the patients was 60 years (range, 39–77), 26/43 (60%) were male, 42/43 (98%) had Binet stage B and C disease, and the median number of prior treatment regimens was 2 (range, 1–4); 32/43 (74%) of patients were relapsed while 11/43(26%) were refractory to prior therapy.Pretreatment included F in 93% of patients (F+C 35%) and monoclonal antibodies (MoAb) in 39.5% of patients (Rituximab 28%, CAM 19%; both MoAb 7%). More than a half of pts presented with deleted 11q or 17p with 28% of pts showing 17p as the sole aberration. Unmutated IgVH was detected in 23/34 (68%) pts while 17/43 (52%) were tested positive for ZAP70. Median number of given cycles was 4. Myelosuppression of all grades was the most commonly occurring adverse events. Grade III-IV neutropenia episodes were observed in 66% of the administered courses while grade III-IV thrombocytopenia episodes were detected only in 8.0 % of cycles. Symptomatic CMV reactivation occurred in 8 cases, no CMV disease was recorded. Twelve major infections occurred, including hepatitis B virus (HBV) reactivation (1 patient), pneumonia (8 episodes), and sepsis (3 episodes). Two major late infections were also observed: 1 tuberculosis and 1 aspergillosis. There were 8 deaths among 29 responding patients due to disease progression (5 patients), Richter's syndrome (2 patients), and one major late infection (tuberculosis). Progressive disease was the cause of death in 11 cases among 14 non-responding patients. An overall response rate (ORR) of 67% (29/43) was recorded with 13 pts (30%) achieving CR (MRD- in 6 pts) and 16 PR (37%). Higher ORR (p=0.003) was observed in pts not previously treated with MoAb. After a median follow up of 16.7 months (range, 1–49.5 mo) patients receiving FCC had a median PFS of 24.4 months. Finalized data of all patients will be presented. Conclusion:Results from the final analysis of this new, 4-weekly dosing FCC regimen suggest that combination therapy with F, C and CAM is effective. Poor prognostic factors were strongly represented in these patients, including 68% unmutated IgVH and 54% deleted 17p or 11q. Although there were a number of major infections, most were manageable. Disclosures:Montillo:Bayer: Research Funding, Speakers Bureau; Genzyme: Research Funding, Speakers Bureau.

Microenvironment Influences Expression of TOSO – a Novel NF-Kappa B Target Gene In Chronic Lymphocytic Leukemia

AbstractAbstract 695We recently identified the transmembrane protein TOSO to be significantly over-expressed in chronic lymphocytic leukemia (CLL) compared to other B-cell lymphomas or healthy B-cells and T-cells. TOSO was initially characterized as inhibitor of Fas-mediator of apoptosis; however, it could be demonstrated to be the receptor for the IgM-specific Fc-domain in immune cells. TOSO is the only Fcμ receptor expressed on B-cells and is solely expressed in the lymphoid compartment. However, little is known on its regulation and the molecular background of over-expression in CLL. We investigated TOSO expression on mRNA and protein level in freshly isolated primary CLL cells (n=10) and healthy B-cells (n=4) after single treatment for 24 hours with a comprehensive panel of different cytokines or stimuli (interleukin (IL)-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, interferon (IFN)-γ, transforming growth factor (TGF)-ß, tumor necrosis factor (TNF)-α, lipopolysaccharide, CpG, CD40-ligand (CD40L) and B-cell receptor (BCR)) being involved in B- and T-cell interplay, by qRT-PCR, western blotting and flow cytometry. Furthermore, we determined the impact of nurse-like cells (NLC) to TOSO expression and co-incubated primary CLL cells for up to 14 days with NLCs. To better understand the intracellular regulation of TOSO, we inhibited BCR and/or CD40L pathways, which were shown by us to be either stimulatory (BCR) or inhibitory (CD40L) in regard to TOSO expression. Since expression might be finally also controlled on epigenetic level, we determined the methylation status of the putative TOSO promoter in 64 CLL samples and 10 healthy B-cells samples. Quantitative DNA methylation analysis was conducted using the EpiTyper application by Sequenom (San Diego, CA, USA). Our experiments reveal novel extra- and intracellular stimuli regulating TOSO expression. We identified CD40L, IL-4 and CpGs to have strong inhibitory effects on TOSO expression (P<0.001) in primary CLL cells and healthy B-cells. In contrast, we identified NLCs (MFIR 15,8 vs. 25,8; P=0.049; n=4) and BCR cross-linking to induce TOSO expression on the cell surface of CLL cells. Based on extracellular stimuli, we were able to hypothesize on shared downstream pathways in order to identify the key regulatory factors and transcription factors controlling TOSO expression. By using a panel of inhibitors in BCR and CD40L downstream signaling, NF-kappa B was shown to have the strongest effect on TOSO expression (P=0,0294). Applying the I-kappa B kinase (IKK) inhibitor Wedelolactone at non-toxic concentrations (10μM), TOSO expression was profoundly suppressed after 24 hours. Regarding epigenetic alterations, our analysis from genome-wide screening experiments in CLL patients compared to healthy B-cells did reveal significant aberrant DNA de-methylation events in the TOSO promoter-associated CpG island (P<0.001). In conclusion, we revealed IL-4, CpG and CD40L as BCR stimulus and NLCs as the key components in regulation of TOSO in the CLL cell microenvironment. Furthermore, over-expression of TOSO in CLL cells compared to normal B-cells could be demonstrated being associated with epigenetic changes at its promoter. We identified TOSO as a novel NF-kappa B regulated target gene. In ongoing studies we elucidate whether NF-kappa B acts directly or in-directly on TOSO expression. Disclosures:No relevant conflicts of interest to declare.

Detection of chronic lymphocytic leukemia cell surface markers using surface enhanced Raman scattering gold nanoparticles

Selective targeting and detection of a hematologic malignancy, chronic lymphocytic leukemia, using surface enhanced Raman scattering (SERS) gold nanoparticles is reported. The functional nanoparticles were composed of a gold core onto which an optical reporter dye was adsorbed, protected from aggregation by grafted polyethyleneglycol, and targeted to CD19 antigen by antibodies. The signals were detected by dark-field microscopy and Raman spectrometry. The observation that the Raman signals are not disrupted by several traditional pathology stains indicates advantages over fluorescence methods.

Receptor Tyrosine Kinase-Like Orphan Receptor 1 (ROR1) - a Putative Diagnostic Marker for Chronic Lymphocytic Leukemia (CLL).

Abstract 1587Poster Board I-613In chronic lymphocytic leukemia (CLL) WNT signaling is constitutively active and several members of this signaling pathway are uniformely upregulated in these cells. Apart from classical WNT receptors like FZD and LRP6, receptor tyrosine kinase-like orphan receptor 1 (ROR1) has been shown to function as a receptor for WNT proteins, too. Furthermore, it could recently be demonstrated that ROR1 is frequently expressed on the surface of CLL cells and might therefore serve as a therapeutic target in this disease. However, so far only little is known about the expression status of this protein in different patients. Moreover, a diagnostic antibody for flow cytometric investigations is lacking. Thus, the aim of our study was to i) establish a directly labelled anti-ROR1 antibody for flow cytometry, ii) to confirm previous results on ROR1 expression in CLL, iii) to investigate ROR1 expression in different cell compartments and iv) correlate our findings to known markers of risk and disease progression. Peripheral blood of CLL patients as well as healthy volunteers was subjected to flow cytometric analysis. Besides standard determination of leukocyte subpopulations ZAP70 and CD38 status was assessed according to current diagnostic recommendations. In addition, ROR1 surface expression was first detected by flow cytometry using a specific primary antibody directed against ROR1 and a fluorescent labelled secondary antibody. Using this experimental setting we found that ROR1 is expressed on 63.4% of all neoplastic CLL cells and also on 30.5% of T cells in the peripheral CLL blood. In contrast, no ROR1 expression could be detected on NK cells, B cells, CD8+- or CD4+-T cells of healthy individuals. To improve the analytical technique the ROR1 antibody was directly conjugated with Phycoerythrin (PE) and the experiments were repeated. With the conjugated antibody we detected ROR1 expression on 97.1% of neoplastic CLL cells and virtually on no T lymphocytes. ROR1 expression levels correlated neither with the expression of ZAP70 nor with CD38. Again, we could not detect ROR1 expression on peripheral blood cells of our healthy volunteers. Taken together, ROR1 expression appears to be highly restricted to CLL cells. If in addition to CD5 and CD19 ROR1 detection is included into diagnostic flow cytometric panels the specificity and sensitivity of immunophenotypic CLL diagnostics may be greatly enhanced. DisclosuresHallek:Roche: Consultancy, Honoraria, Research Funding.

Targeted therapy for chronic lymphocytic leukemia.

The introduction of targeted agents such as the monoclonal antibodies rituximab (anti-CD20) and alemtuzumab (anti-CD52) has brought about a remarkable change in the therapy of chronic lymphocytic leukemia (CLL). Although it is unclear whether overall survival has been improved, the incorporation of these monoclonal antibodies into chemoimmunotherapy regimens has dramatically improved complete response rates and progression-free survival in patients with both newly-diagnosed and relapsed CLL. The success of rituximab and alemtuzumab has spurred the development of other monoclonal antibodies targeting distinct proteins and epitopes on the surface of CLL cells and an exciting array of novel immunotherapeutics. The judicious use of these agents provides the opportunity to develop risk-adapted therapeutic strategies to optimize responses and quality of life in patients with CLL.

High Incidence of Autoimmune Complications during Combined Fludarabine and Rituximab Therapy in CLL: Rapid Clearance of Rituximab as a Contributing Factor

Autoimmune cytopenias (AIC), particularly autoimmune hemolytic anemia (AIHA), pure red cell aplasia (PRCA) and immune-thromobcytopenic purpura (ITP) are important complications of fludarabine (F) treatment of chronic lymphocytic leukemia (CLL). Rituximab (R), a chimeric anti-CD20 monoclonal antibody, is an effective treatment for these autoimmune complications. The introduction of rituximab into fludarabine containing combination regimens has been expected to decrease the incidence of AIC. The experience in some studies (CALBG 9712, 2% incidence of AIC with FR; Byrd, 2003) but not others (6.5% incidence in 300 patients treated with FCR; Borthakur, 2007) supported this view. We compared the incidence of AIC during treatment in 2 CLL cohorts, one treated with F (1998–2004, n=21), the other with FR (2005–2008, n=17, rituximab 375mg/m2 q4 weeks). Five of 21 patients (24%) in the F group developed AIC (3 ITP, 1 PRCA, 2 AIHA), while 3 of 17 patients (18%) in the FR group developed AIC (2 AIHA, 1 autoimmune neutropenia). Two patients with ITP in the first cohort and all three patients in the second were successfully treated for their AIC with 4 infusions of rituximab over 2–4 weeks. Interestingly, although rituximab did not prevent the occurrence of AIC it remained effective in treating the complication, indicating that serum levels of rituximab could be too low to prevent the onset of AIC. We therefore measured rituximab serum levels in 7 patients (average ALC 134 K/mL, range 10–423) at 6, 24 and 120 hours from the start of the infusion. The average peak serum level at 6 hours, typically shortly after the end of the infusion, was 93μg/mL (32–155). By 24 hours, the average level declined to 66μg/mL (39–90), and by 120 hours to 11μg/mL (0–30, undetectable in 2). Based on these measurements, we estimate the half life of rituximab during this first cycle at 1–2 days. This extremely short half life is consistent with enhanced clearance of cell bound rituximab either during phagocytosis proper or during a form of abortive phagocytosis, also called trogocytosis, during which rituximab/CD20 complexes are pulled off the CLL cell surface and ingested by phagocytic cells (Beum, 2006). Consistent with the expected correlation of high tumor burden with rituximab clearance, we found higher rituximab serum levels in subsequent cycles: in cycle 2, 3 of 4 patients had an average increase in serum concentration of 27% at 24 hours, and in cycle 3 one patient had a 72% higher level than in the first cycle. Thus, at least in the first cycles, rituximab is cleared within a week, giving rise to a prolonged period of essentially single agent fludarabine effects. This could contribute to the persistently high incidence of AIC despite the use of rituximab in fludarabine combination regimens. These data indicate that, at least in the first cycles, additional infusions of rituximab may be necessary to prevent the onset of AIC in CLL patients treated with fludarabine-containing regimens.

Chronic lymphocytic leukemia (CLL) cells genetically modified to express B7-1, ICAM-1, and LFA-3 confer APC capacity to T cells from CLL patients

In chronic lymphocytic leukemia (CLL), malignant B cells and nonmalignant T cells exhibit dysfunction. We previously demonstrated that infection of CLL cells with modified vaccinia Ankara (MVA) expressing the costimulatory molecules B7-1, ICAM-1, and LFA-3 (designated TRICOM) increased expression of these costimulatory molecules on the surface of CLL cells and thus augmented their antigen-presenting capability. Here, we evaluate the effect of MVA-TRICOM-modified CLL cells on T cells. Following incubation with irradiated MVA-TRICOM-modified CLL cells, allogeneic and autologous CD4(+) and CD8(+) T cells expressed significantly higher levels of B7-1, ICAM-1, and LFA-3. We show that this increase was the result of physical acquisition from the antigen-presenting cells (APCs), and that purified T cells that acquired costimulatory molecules from MVA-TRICOM-modified CLL cells were able to stimulate the proliferation of untreated T cells. These results demonstrate for the first time that T cells from CLL patients can acquire multiple costimulatory molecules from autologous CLL cells and can then act as APCs themselves. Given the immunodeficiencies characteristic of CLL, enhancing the antigen-presenting function of CLL cells and T cells simultaneously could be a distinct advantage in the effort to elicit antitumor immune responses.

Free Light Chains, Monoclonal Proteins, and Chronic Lymphocytic Leukemia.

Monoclonal proteins(MPs) can frequently be detected in the serum/urine of chronic lymphocytic leukemia(CLL) patients. Serum free light chains(FLC) assays can detect MPs in the absence of M bands on immunofixation(IMF).An abnormal FLC ratio indicates excess of one light chain type suggesting clonality.We evaluated fresh serum/urine samples from 34 CLL patients at various stages of disease by quantitative nephelometric assay,IMF and FLC assay.Median age was 66 yrs(43–87),M:F 1.9:1 and median time from diagnosis 41.5 mos(5–288). 45% had advanced stage and 39% prior treatment with 1–7 therapies.Only 2 patients had mild renal failure.Serum immunoglobulins were normal/low in 94% of patients:IgG (171–1580 mg/l, median 803 mg/l), IgA(<25–310 mg/l, median 88 mg/l),IgM(<18–290 mg/l, median 38 mg/l), irrespective of the presence of a monoclonal protein. 71% of patients had evidence of abnormal immunoglobulin synthesis: by IMF alone (8),IMF and FLC(10) or FLC alone(8).Abnormal FLC ratios were more frequently associated with advanced stage disease and increased κ chains(see table 1). Abnormal FLC ratios(< 0.26 or >1.65) were measured in 18(53%) patients, 8 of whom did not have MPs by IMF. Two advanced stage patients had abnormal FLC ratios due to very low levels of a single light chain, most probably reflecting secondary hypogammaglobulinemia due to CLL and/or chemotherapy.Abnormal FLC ratios reverted to normal after 1 course of chemotherapy(FC+/−R) in 3 patients despite persistence of minimal residual disease(MRD).FLC were of the same type as expressed on the surface of CLL cells, with discrepancies observed in 5 patients.BM biopsy staining for light chains revealed IgAκ MGUS in addition to λ chain restricted CLL in one patient, but was noncontributory in the other 4.Conclusions: Neither IMF nor the FLC assay alone could detect all MPs. Normal FLC ratios do not exclude the presence of MPs. The FLC ratio should be interpreted with caution in CLL patients with advanced disease and hypogammaglobulinemia. Monoclonal bands and abnormal FLC ratios can be detected despite normal or low levels of serum immunoglobulins in CLL patients. The significance of these monoclonal gammopathies in CLL is not clear and warrants further study in a larger group of patients. Discrepancies between surface immunoglobulins and serum/urine MPs might suggest the presence of an additional condition:ex. MGUS. FLC may not be a sensitive measure of MRD.Normal FLC ratioAbnormal FLC ratioP valueNo. patients16(47%)18(53%)M:F1.3:13.5:1NSAge*62.5 yrs(49–73)68 yrs(43–87)NSTime from CLL diagnosis*25.3 mos(0–282)59 mos(0–210)NSAdv stage(Rai III/IV-Binet C)25%66.7%0.018Untreated62%50%NSCLL:κ:λ restriction7:8(0.88:1)11:6(1.8:1)NSMonoclonal protein8(50%)10(56%)NSIncreased freeκ no.8150.043Freeκ level*19.6(4.5–200)39.2(2.9–386)0.0064Increased freeλ no43NSFree λ level*19.3(12.4–146)15.4(0.1–517)NSSerum β 2m*3.1(1.8–6.7)4.3(2.2–10.2)NSZap70≥2086%36%0.002CD38≥3044%44%NS17pdel/11qdel40%44%NS*median

Activation of Nucleic Acid-Sensing Toll-Like Receptors Induces Proliferation, Cytokine Production, Immunogenic Phenotype, and Plasma Cell Differentiation of CLL Cells and Immunoglobulin Production.

Weak immune function of chronic lymphocytic leukemia (CLL) cells may contribute to disease progression and inhibit effective immunotherapy. Accordingly, agents that enhance the function of CLL cells may be useful in immunotherapeutic approaches to this disease. Since Toll-like receptors (TLRs) are major regulators of B-cell function in innate and adaptive immunity, we asked to what extent stimulating two nucleic acid-sensing TLRs affected the activation and differentiation of CLL B cells. Isolated CLL cells were treated with either the TLR7-activating ligand, gardiquimod, or the TLR9-activating ligand ODN 2006-G5. CLL cells were also treated with the contents of apoptotic leukemic B cells, produced by repeated freezing and thawing. Ability to induce proliferation, as defined by CFSE dilution studies, cytokine production, differentiation, and altered surface molecule expression was measured. Both TLR 7 and TLR 9 ligands individually induced high levels of proliferation and plasma cell differentiation of CLL B cells. A dramatic increase in percentage of dividing cells was seen with simultaneous TLR7 and TLR9 activation, while blocking the TLR 7/9 pathway by pre-treating CLL B cells with chloroquine or bafilomycin reduced the proliferative capacity by 90%. Furthermore, stimulating CLL B cells with TLR7 and 9 ligands significantly up-regulated activation (CD38, CD69 and CD83), adhesion (CD54), and other immunophenotypic (CD40, CD80 and CD86) markers. Enzyme immunoassays of culture supernatants after TLR 7 and TLR 9 stimulation indicated that the levels of soluble IgM increased considerably, although levels of cytoplasmic IgM were unchanged. Production of IL6 and IL10 was also induced. We propose that ligands for TLRs 9 and 7 stimulate CLL B cells to proliferate and differentiate into plasma cells in the absence of T-cell help. In addition these stimuli alter CLL cell surface phenotype to a more immunogenetic profile, providing a rationale for CpG oligonucleotides and gardiquimod as humoral vaccine adjuvants for both normal immune reactions and potentially for anti-leukemic therapy.